Method of detecting specific polymer crystal

ABSTRACT

In a specific macromolecule crystal detecting method according to the present invention, ultraviolet light is irradiated to sample solution, and a fluorescent image emitted from a sample in the sample solution is detected to detect specific macromolecules in the sample solution. Furthermore, by detecting the outline of the sample from the visible light image of the sample contained in the sample solution, the crystal is discriminated from other materials on the basis of the outline. By integrating the detection results of the fluorescent image and the visible light image, the specific macromolecule crystal is detected from the sample solution.

BACKGROUND OF THE INVENTION

The present invention relates to a detection method of detecting aspecific polymer crystal, namely a specific macromolecule crystal insample solution.

Worldwide attention has been paid to the structural analysis of proteincrystals in connection with the development in the genome plane since adouble helix structure of DNA was discovered. A method using NMR(Nuclear Magnetic Resonance apparatus) a method using an electronmicroscope, a method using the X-ray diffraction phenomenon, etc. havebeen developed for the structure analysis of protein crystals, andparticularly the X-ray crystal structure analysis using the X-raydiffraction phenomenon has been rapidly advanced in connection with thedevelopments of a two-dimensional X-ray detector such as an imagingplate or the like, analyzing software for two-dimensional data, etc.

In the protein crystal structure analysis using the X-ray diffractionphenomenon, it has been hitherto general that a protein crystal grainachieved by crystallizing protein in solution is first poured into anarrow tube called as a capillary together with the solution, and thecapillary concerned is mounted in an X-ray diffraction device.

In order to carry out the X-ray structural analysis of protectioncrystals by using the X-ray diffraction deice as described above, aprotein crystal as a target must be accurately positioned to an X-rayirradiating position. Therefore, after a sample solution pouredcapillary is mounted in the X-ray diffraction device, the proteincrystal in the capillary is visually observed by using a microscope, andit is manually positioned to the X-ray irradiating position.

The search of the protein crystal through the visual observation asdescribed above and the positioning work are cumbersome and take muchtime. In addition, these works must be carried out every time onemeasuring operation is finished, and thus this method is unsuitable torapidly measure/evaluate many samples automatically.

The number of proteins constituting a human body extends to 50,000 to100,000 kinds of proteins, and it has been urgently required in therecent structural biology to clarify many protein structures in a shorttime.

The present invention has been implemented in view of the foregoingsituation, and has an object to provide a specific macromolecule crystaldetecting method that can easily search a specific macromolecule crystalsample from sample solution and contribute to the quick processing ofmeasuring/evaluating specific macromolecule crystals.

SUMMARY OF THE INVENTION

In order to attain the above object, a specific macromolecule crystaldetecting method according to the present invention is characterized inthat a sample in a sample container is identified as a specificmacromolecule under the condition that the sample in the solutiongenerates fluorescence when ultraviolet light is irradiated to thesample solution, and it is judged on the basis of a visible light imageof the sample whether the sample is a crystal or not.

Furthermore, a specific macromolecule crystal detecting methodcomprises:

a specific macromolecule detecting step of irradiating ultraviolet lightto sample solution and detecting a fluorescent image emitted from asample in the sample solution; and

a crystal detecting step of detecting the outline of the sample from thevisible light image of the sample contained in the sample solution,wherein a sample for which a fluorescent image is detected in thespecific macromolecule detecting step and an outline indicating acrystal is detected in the crystal detecting step is identified as aspecific macromolecule crystal.

Most of polymer crystals, particularly biological polymer generatesfluorescence when ultraviolet light is irradiated thereto. In thisspecification, the polymer crystal having a characteristic that itgenerates fluorescence when ultraviolet light is irradiated to thepolymer crystal will be referred to as “specific macromolecule crystal”.For example, the protein crystals correspond to the specificmacromolecule crystals.

According to the specific macromolecule crystal detecting method of thepresent invention, paying attention the characteristic of the specificmacromolecule crystal as described above, ultraviolet light isirradiated to sample solution and a fluorescent image emitted from asample in the sample solution is detected, thereby detecting specificmacromolecule in the sample solution.

However, there is a case where it is unidentifiable on the basis of onlythe fluorescent image whether the detected specific macromolecule formsa crystal or not. For example, when aggregation of specificmacromolecule exists in sample solution, the aggregation concernedgenerates fluorescence, and thus a fluorescent image of the crystal anda fluorescent image of the aggregation are detected with being mixedwith each other.

Therefore, according to the specific macromolecule crystal detectingmethod of the present invention, the outline of the sample is detectedon the basis of a visible light image of the sample contained in thesample solution to discriminate the crystal from materials other thanthe crystal on the basis of the outline, and the “crystal” of “specificmacromolecule” is detected from the sample solution in cooperation withthe detection result of the fluorescent image.

Furthermore, the specific macromolecule crystal according to the presentinvention further comprises a step of recognizing the position of thesample identified as a specific macromolecule crystal in addition to theabove construction.

Furthermore, the crystal detecting step may comprise:

an image input step for achieving the visible light image of the samplecontained in the sample solution as image data;

an image processing step for binarizing the image data of the visiblelight image thus achieved;

an edge detecting step of detecting pixels corresponding to an edge ofthe sample contained in the sample solution from the binarized imagedata;

a contour line detecting step of searching continuity of the pixelscorresponding to the edge of the detected sample and detecting a closedcontour line of the sample; and

a gravity center detecting step of recognizing an internal area of theclosed contour line and detecting the position of the center of gravityof the internal area.

A sample having a closed contour line recognized in the crystaldetecting step is evaluated as a crystal having a constant area, and asample whose contour line is not closed is evaluated as anon-crystallized sample such as aggregation or the like. Therefore,attention is paid to only the sample having the closed contour line, andthe gravity center position of the sample concerned is detected. It canbe judged by integrating the identification result of the specificmacromolecule detecting step whether the sample is a specificmacromolecule crystal or not.

The specific macromolecule crystal detecting method according to thepresent invention can perform automatic processing based on computerprocessing. Accordingly, it can be utilized by automating themeasurement/evaluation of specific macromolecule crystal samples, and itis expected to contribute to the rapid processing of themeasurement/evaluation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the scheme of a protein crystal detectingdevice suitable to execute the method of the present invention.

FIG. 2A is a perspective view showing a construction of a samplecontainer.

FIG. 2B is a partially enlarged front cross-sectional view showing thesample container.

FIG. 3 is a flowchart showing a protein crystal detecting methodexecuted by a central processing unit.

FIG. 4 is a flowchart showing a subroutine according to step S4 of FIG.3.

FIG. 5 is a diagram showing the edge detecting processing of step S11shown in FIG. 4.

FIG. 6 is a diagram showing the scheme of a protein crystal evaluatingdevice in which the specific macromolecule detecting device of FIG. 1 isinstalled.

FIG. 7A, 7B are sketches of microscope images achieved by observingsample solution in which a protein crystal and a crystal of materialgenerating self-fluorescence are mixed.

FIG. 8A, 8B are sketches of microscope images achieved by observingsample solution in which aggregation of protein is contained.

DETAILED DESCRIPTION OF THE INVENTION

Preferred embodiments according to the present invention will bedescribed with reference to the drawings. In the following embodiments,the description will be made on the assumption that a protein crystal isset as a detection target (a specific macromolecule crystal).

FIG. 1 is a diagram showing the scheme of a protein crystal detectingdevice suitable to execute the method of the present invention.

The protein crystal detecting device shown in FIG. 1 is equipped with avisible light irradiating unit 2 and a ultraviolet light irradiatingunit 3 at the lower side of a sample table 1. The visible lightirradiating unit 2 and the ultraviolet light irradiating unit 3 arelight sources for irradiating visible light or ultraviolet light tosample solution L in a sample container 10 disposed on the sample table1.

The visible light irradiating unit 2 and the ultraviolet lightirradiating unit 3 are laterally slid so that any one of them isdisposed so as to confront the sample container 10 on the sample table 1through a through hole 1 a. If a reflection mirror is disposed at themidpoint between the sample container 10 and each of the visible lightirradiating unit 2 and the ultraviolet light irradiating unit 3 so thatvisible light emitted from the visible light irradiating unit 2 orultraviolet light emitted from the ultraviolet light irradiating unit 3is led to the sample container 10, it is unnecessary each of theirradiating units 2, 3 is disposed so as to confront the samplecontainer 10.

The sample table 1 is provided with a through hole 1 a through whichvisible light irradiated from the visible light irradiating unit 2 andultraviolet light irradiating from the ultraviolet light irradiatingunit 3 are transmitted, and the sample container 10 is mounted on thethrough hole.

It is preferable that a crystallization plate formed of material such aspolyimide or the like through which ultraviolet light, visible light andX-ray are transmitted is used for the sample container 10. As shown inFIG. 2A, many recess portions 11 are formed on the sample container 10using the crystallization plate, and protein crystals can be generatedin the recess portions 11. Various methods containing a vapor diffusionmethod are known as a method of generating a protein crystal by using acrystallization plate. FIG. 2B is a schematic diagram showing a statewhere a protein crystal S is generated by the vapor diffusion method,and a protein crystal S is generated in a drop of sample solution Lplaced on the lower surface of a cover plate 12. Protein crystals areseparately generated in the many recess portions formed on the samplecontainer while the generation condition is varied, or different kindsof protein crystals can be separately generated therein.

Returning to FIG. 1, a microscope 4 and a two-dimensional image pickupunit 5 are disposed above the sample table 1. The microscope 4 enlargesan image achieved when ultraviolet light or visible light is irradiatedto the sample solution L in the sample container 10 and then transmittedthrough the sample solution L, and leads the enlarged image to thetwo-dimensional image pickup unit 5. The microscope 4 may be designed sothat the protein crystal S in the sample solution L can be searched byvarying the focal position in the vertical direction.

For example, CCD may be used as the two-dimensional image pickup unit 5.The two-dimensional image pickup unit 5 converts the enlarged imageincident through the microscope 4 to an electrical signal (image data),and outputs the electrical signal to the central processing unit (CPU)6. The central processing unit 6 processes the image data input from thetwo-dimensional image pickup unit 5 to detect the protein crystal S inthe sample solution L and also recognize the position thereof.

Next, a protein crystal detecting method using the above device will bedescribed.

FIGS. 3 and 4 are flowcharts showing the protein crystal detectingmethod executed by the central processing unit.

First, the sample container 10 in which the protein crystal S isgenerated in the sample solution L is disposed on the through hole 1 aof the sample table 1. Subsequently, a light source is set to theultraviolet irradiating unit 3, and ultraviolet light emitted from theultraviolet light irradiating unit 3 is irradiated to the samplesolution L in the sample container 10.

At this time, an image achieved when the light is transmitted throughthe sample solution L in the sample container 10 is enlarged by themicroscope 4 and then incident to the two-dimensional image pickup unit5. The central processing unit 6 receives the image data transmittedfrom the two-dimensional image pickup unit 5 (step S1), and detects thefluorescent image from the image data (step S2). That is, the proteincrystal S generated in the sample solution L generates fluorescence whenultraviolet light is irradiated thereto, and thus the fluorescent imagethereof is incident to the two-dimensional image pickup unit 5.Therefore, the central processing unit 6 analyzes the image data inputfrom the two-dimensional image pickup unit 5 to detect the fluorescentimage and grasp the position of the fluorescent image, that is, theprotein. The position of the protein thus grasped corresponds to aposition on the horizontal plane (XY coordinate), and the position inthe height direction (z coordinate) is grasped on the basis of the focalposition of the microscope 4.

Subsequently, the light source is switched from the ultraviolet lightirradiating unit 3 to the visible light irradiating unit 2, andirradiates the visible light emitted from the visible light irradiatingunit 2 to the sample solution L in the sample container 10. At thistime, a visible light image achieved through the sample solution L inthe sample container 10 is enlarged by the microscope 4 and thenincident to the two-dimensional image pickup unit 5. The centralprocessing unit 6 receives the image data transmitted from thetwo-dimensional image pickup unit 5 (step S3), and processes the imagedata to detect a crystal in the sample solution L and also recognize theposition of the gravity center of the crystal (step S4).

The step S4 (crystal detecting step) is processed along a subroutineshown in FIG. 4. That is, the image data input from the two-dimensionalimage pickup unit 5 is subjected to binarization processing by using apredetermined threshold value as a reference, and converts each pixel onthe xy coordinate to binary data of “1” or “0” (step S10).

Subsequently, the pixels corresponding to the edge of a sample existingin the sample solution are detected on the basis of the binarized imagedata (step S11). In this case, for example it is judged whether a notedpixel as a identification target is black (data “1”) as shown in FIG. 5,and if it is black, it is identified for the surrounding pixels (pixels1 to 8) of the noted pixel concerned whether each of them is black (data“1”) or white (data “0”).

If all the surrounding pixels (pixels 1 to 8) are white (data “0”), itis concluded that the noted pixel is an isolated point. If all thesurrounding pixels (pixels 1 to 8) are black (data “1”), it is concludedthat the noted pixel concerned is an internal point of an image. Asdescribed above, all the pixels corresponding to isolated points andinternal points are excluded, and a noted pixel for which some ofsurrounding pixels (pixels 1 to 8) of the noted pixel concerned arewhite (data “0”) is recognized as an edge of the sample, and the xycoordinate of the noted pixel concerned is stored.

The above processing is executed on all the pixels on the xy coordinatesystem, and all the pixels corresponding to the edge of the sample areextracted.

Subsequently, the pixels corresponding to the edge of the sample thusextracted are noted, and the neighboring pixels thereof are linked toone another to detect the contour line of the sample (step S12). If thestart and end points of the contour line are coincident with each other,the contour line is identified as a closed contour line. The samplehaving the closed contour line is identified as a crystal having aconstant area. On the other hand, the sample whose contour line is notclosed is excluded as a non-crystallized material such as aggregation orthe like.

Subsequently, the internal area of the sample having the closed contourline (that is, crystal) is recognized, and the gravity center positionof the internal area is calculated by a well-known calculation method(step S13).

As a method of calculating the gravity center position of a planarimage, for example, the moment quantity of a linked figure S recognizedas a crystal is calculated, and the gravity center position iscalculated from this moment quantity. That is, when the weight of eachpixel of the linked figure S is equally set to 1, the moment M(m,n) isdefined by the following equations.

${M\left( {m,n} \right)} = {\sum\limits_{{({x,y})} \in S}\left( {x^{m} \times y^{n}} \right)}$

M(0, 0) represents the area of the linked figure S

M(1,0) represents the moment with respect to the x-axis

M(0,1) represents the moment with respect to the y-axis

The gravity center coordinate (p, q) can be calculated by using theabove moment quantity according to the following equations:P=M(1,0)/M(0,0)Q=M(0,1)/M(0,0)

After the gravity center position of the crystal thus detected iscalculated, the central processing unit 6 returns to the main routineshown in FIG. 3 again, and superposes the position of the proteindetected on the basis of the fluorescent image with the position of thecrystal detected on the basis of the visible light image to recognizethe protein crystal S. The gravity center position achieved n step S13of FIG. 4 for the protein crystal S is stored (step S5). As describedabove, the gravity center position of the protein crystal S existing inthe sample container 10 can be automatically detected.

FIG. 6 is a diagram showing the scheme of a protein crystal evaluatingdevice in which a specific macromolecule detecting device is installed.

The specific macromolecule detecting device 20 can constitute a devicefor automatically evaluating a protein crystal in combination with anX-ray diffraction apparatus 30. That is, the sample table 1 of thespecific macromolecule detecting device 20 and a sample table 31 of theX-ray diffraction apparatus 30 are connected to each other by movingmeans 40 such as an X-Y table or the like, and the sample container 10is automatically fed from the specific macromolecule detecting device 20to the X-ray diffraction apparatus 30, whereby evaluation of the proteincrystal S can be automatically carried out by the X-ray diffractionapparatus 30.

As well known, the X-ray diffraction apparatus 30 is equipped with anX-ray source 32 and an X-ray detector 33, and when X-ray emitted fromthe X-ray source 32 is irradiated to the protein crystal S, diffractedX-ray emitted at a predetermined diffraction angle is detected by theX-ray detector 33. The diffraction angle at which the diffracted X-rayis emitted is determined by the crystal structure of material, and thusthe structure of a protein crystal can be evaluated on the basis of thediffraction angle.

Here, in order to execute the evaluation of the protein crystal by theX-ray diffraction apparatus 30, it is necessary to accurately positionthe protein crystal to the X-ray irradiation position. Therefore, first,the gravity center position of the protein crystal S existing in thesample container 10 is detected by using the specific macromoleculedetecting device 20, and the moving means 40 is controlled to feed thesample container 10 so that the gravity center position concerned ispositioned to the X-ray irradiating position of the X-ray diffractionapparatus 30.

By using the protein crystal evaluating device thus constructed, aprotein crystal generated in each recess portion 11 of thecrystallization plate as shown in FIG. 2 can be automaticallymeasured/evaluated, and the working can be facilitated and the speed-upof the measurement/evaluation can be implemented.

EXAMPLES

FIGS. 7A and 7B are sketches of microscope images achieved by observingsample solution in which a protein crystal and a crystal of materialgenerating no self-fluorescence were mixed, wherein FIG. 7A shows avisible light image achieved by irradiating visible light to the samplesolution, and FIG. 7B shows a fluorescent image achieved by irradiatingultraviolet light to the sample solution.

As shown in FIG. 7A, when visible light was irradiated to the samplesolution, a visible light image A of the protein crystal and a visiblelight image B of another crystal were observed. It is unidentifiable onthe basis of the above image which one of the visible light imagescorresponds to the protein crystal.

However, as shown in FIG. 7B, when ultraviolet light was irradiated tothe sample solution, only a fluorescent image C of the protein crystalwas observed, and the other crystal was not detected. Accordingly, bysuperposing the visible light image A and the fluorescent image C oneach other, the position of the protein crystal could be recognized.

FIGS. 8A, 8B are sketches of microscope images achieved by observing thesample solution in which aggregation of protein was contained, whereinFIG. 8A is a fluorescent image achieved by irradiating ultraviolet lightto the sample solution, and FIG. 8B is a visible light image achieved byirradiating visible light to the sample solution.

As shown in FIG. 8A, when ultraviolet light was irradiated to the samplesolution, a fluorescent image D emitted from the aggregation of proteinwas observed. It is unidentifiable on the basis of the above fluorescentimage D whether it is the aggregation of the protein or the crystal ofthe protein.

However, as shown in FIG. 8B, when visible light is irradiated to thesample solution, in the case of aggregation of protein, no clear ridgeline of a crystal is observed, and it is discontinuous or have aneedle-like outline in many cases. Accordingly, on the basis of thisfact, it can be judged whether the observation target is aggregation ofprotein or the like.

As described above, by integrating the fluorescent image achieved whenultraviolet light is irradiated to sample solution and the visible lightimage achieved when visible light is irradiated to the sample solution,the position of the protein crystal can be recognized with excludingcrystals other than the protein crystal and aggregation of protein.

In the above embodiments and examples, the description has been made bysetting the protein crystal as a detection target, however, the targetof the method of the present invention is not limited to the proteincrystal. Various kinds of specific macromolecule crystals having thecharacteristic that they generates fluorescence when ultraviolet lightis irradiated thereto can be set as detection targets.

As described above, according to the present invention, the specificmacromolecule crystal can be easily searched by integrating thefluorescent image achieved when ultraviolet light is irradiated tosample solution and a visible light image achieved when visible light isirradiated to the sample solution.

1. A specific macromolecule crystal detecting method comprising: aspecific macromolecule detecting step in which a sample solution isirradiated with irradiating ultraviolet light and a fluorescent imageemitted from a sample in the sample solution is detected; and a crystaldetecting step in which the outline of the sample is detected from avisible light image of the sample contained in the sample solution, thecrystal detecting step itself includes the steps of: an image input stepfor obtaining a visible light image of the sample contained in thesample solution as image data; an image processing step for binarizingthe image data of the obtained visible light image; an edge detectingstep of detecting pixels corresponding to an edge of the samplecontained in the sample solution from the binarized image data; acontour line detecting step of searching continuity of the pixelscorresponding to the detected edge of the sample and detecting a closedcontour line of the sample; and a gravity center detecting step ofrecognizing an internal area of the closed contour line and detectingthe position of the center of gravity of the internal area; wherein asample for which a fluorescent image is detected in the specificmacromolecule detecting step and an outline indicating a crystaldetected in the crystal detecting step is identified as a specificmacromolecule crystal.
 2. A specific macromolecule crystal detectingmethod comprising: a specific macromolecule detecting step in which asample solution is irradiated with irradiating ultraviolet light and afluorescent image emitted from a sample in the sample solution isdetected; and a crystal detecting step in which the outline of thesample is detected from a visible light image of the sample contained inthe sample solution, the crystal detecting step itself includes thesteps of: an image input step for obtaining a visible light image of thesample contained in the sample solution as image data; an imageprocessing step for binarizing the image data of the obtained visiblelight image; an edge detecting step of detecting pixels corresponding toan edge of the sample contained in the sample solution from thebinarized image data; a contour line detecting step of searchingcontinuity of the pixels corresponding to the detected edge of thesample and detecting a closed contour line of the sample; and a gravitycenter detecting step of recognizing an internal area of the closedcontour line and detecting the position of the center of gravity of theinternal area; identifying the sample for which a fluorescent image isdetected in the specific macromolecule detecting step and an outlineindicating a crystal is detected in the crystal detecting step as aspecific macromolecule crystal; and recognizing the position of thesample identified as a specific macromolecular crystal.
 3. A specificmacromolecule crystal detecting method comprising: a specificmacromolecule detecting step in which a sample solution is irradiatedwith ultraviolet light from a UV light source causing at least a portionof the sample to fluoresce creating a fluorescence image of the samplein the sample solution that is captured by a two-dimensional imagepick-up unit that converts the fluorescence image to fluorescence imagedata and transmits that data to a CPU; a crystal detecting step in whichthe closed-contour outline of the sample is detected from a visiblelight image of the sample contained in the sample solution, the crystaldetecting step itself includes the steps of: an image input stepcomprising irradiating the sample in the sample solution with visiblelight to create a visible light image of the sample in the samplesolution that is captured by the two-dimensional pick-up unit thatconverts the visible light image to visible light image data andtransmits that image data to the CPU; an image processing step ofbinarizing the visible light image data in the CPU; an edge detectingstep of detecting pixels corresponding to the edge of the samplecontained in the sample solution from the binarized image datatransmitted to the CPU; a contour line detecting step of searchingcontinuity of the pixels corresponding to the detected edge of thesample and detecting a closed contour line of the sample in the CPU; anda gravity center detecting step of recognizing an internal area of theclosed contour line and calculating the position of the center ofgravity of the internal area in the CPU; wherein a sample for which afluorescent image is detected in the specific macromolecule detectingstep and an outline indicating a crystal detected in the crystaldetecting step is identified as a specific macromolecule crystal.
 4. Aspecific macromolecule crystal detecting method comprising: a specificmacromolecule detecting step in which a sample solution is irradiatedwith ultraviolet light from a UV light source causing at least a portionof the sample to fluoresce creating a fluorescence image of the samplein the sample solution that is captured by a two-dimensional imagepick-up unit that converts the fluorescence image to fluorescence imagedata and transmits that data to a CPU; a crystal detecting step in whichthe closed-contour outline of the sample is detected from a visiblelight image of the sample contained in the sample solution, the crystaldetecting step itself includes the steps of: an image input stepcomprising irradiating the sample in the sample solution with visiblelight to create a visible light image of the sample in the samplesolution that is captured by the two-dimensional pick-up unit thatconverts the visible light image to visible light image data andtransmits that image data to the CPU; an image processing step ofbinarizing the visible light image data in the CPU; an edge detectingstep of detecting pixels corresponding to the edge of the samplecontained in the sample solution from the binarized image datatransmitted to the CPU; a contour line detecting step of searchingcontinuity of the pixels corresponding to the detected edge of thesample and detecting a closed contour line of the sample in the CPU; anda gravity center detecting step of recognizing an internal area of theclosed contour line and calculating the position of the center ofgravity of the internal area in the CPU; identifying the sample forwhich a fluorescent image is detected in the specific macromoleculedetecting step and an outline indicating a crystal is detected in thecrystal detecting step as a specific macromolecule crystal; andrecognizing the position of the sample identified as a specificmacromolecular crystal.